This invention relates to an improved process for the extraction, isolation, and purification of secoisolariciresinol diglycoside (SDG) from de-fatted flaxseed.
Flaxseed is presently grown for its oil content, used primarily as an industrial oil. The scientific literature contains an abundance of reports on the chemistry and physical properties of various components derived from de-fatted flaxseed. Recently, considerable interest has been demonstrated in a class of minor compounds contained in flaxseed collectively referred to as lignans.
Lignans are generally dimers containing a dibenzylbutane skeleton. When part of the human diet, these compounds are believed to be converted into the mammalian lignans known as enterolactone and enterodiol. (Thompson et al. xe2x80x9cMammalian Lignan Production from Various Goodsxe2x80x9d, Nutr. Cancer 16:43-52, (1991).) The principal lignan found in flaxseed is secoisolariciresinol diglucoside, referred to hereinafter as SDG.
Flaxseed typically contains 40% by weight fat (xe2x80x9clinseed oilxe2x80x9d). De-fatted (hexane-extracted) flax seed contains a residue of about 2% by wt. fat, with the remainder comprising: 46% by wt. fiber (both water-soluble fiber or xe2x80x9cmucilage,xe2x80x9d acidic heterogeneous polysaccharides that contain galacturonic acid, galactose, rhamnose, and xylose, comprising 30-40% of the total fiber present, and water-insoluble fiber, which comprises 60-70% of the total fiber present); 10% total other carbohydrates, including lignans; 35% by wt. protein; 6-7% ash.
Appreciable amounts of free SDG do not occur in the de-fatted flaxseed. This compound must be liberated by alkaline hydrolysis of various ester-linked polymers. According to the literature, the available SDG in de-fatted flaxseed ranges from 0.9% to 3.0% by wt. (Thompson et al. xe2x80x9cMammalian Lignan Production from Various Goodsxe2x80x9d, Nutr. Cancer 16:43-52, (1991).)
There is considerable published evidence indicating that lignans as a class of compounds exhibit a broad spectrum of biological activities, including anti-tumor, anti-mitotic, anti-oxidant, anti-viral, weak estrogenic and anti-estrogenic activities. Studies conducted by the chemotherapy program of the National Cancer Institute indicate that some lignans prevent the growth of tumors. (Thompson et al, xe2x80x9cAnticarcinogenic Effect of a Mammalian Lignan Precursor from Flaxseedxe2x80x9d, Proc. 55th Flax Institute of U.S.A., Fargo, N. Dak., 46-50 (1194).)
Even though there would appear to be very significant commercial uses for lignans like SDG in food supplements, nutraceuticals, and medicines, SDG has remained a laboratory curiosity, principally because it is available in only very limited quantities. To date, only one commercial process has been developed for the extraction and isolation of SDG from flaxseed. Disclosed herein is an improved process for extracting and isolating SDG from de-fatted flaxseed in order to make the compound available less expensively not only in large quantities, but at a purity suitable for use as a nutritional supplement or nutraceutical.
In 1956 Bakke and Klosterman described a laboratory process for extracting SDG from defatted flaxseed using equal parts of 95% ethanol and 1,4-dioxane. (Bakke and Klosterman, xe2x80x9cA New Diglucoside from Flaxseedxe2x80x9d, Proceedings of the N. Dakota Academy of Science 10:18-22 (1956).)
Prior publications also refer to methanolysis of complexed SDG (i.e. the ester-linked polymers) and to the use of a sodium or barium methoxide for methanolysis to release non-complexed SDG.
U.S. Pat. No. 5,705,618 issued to Wescott et al, entitled xe2x80x9cProcess for Extracting Lignans from Flaxseed,xe2x80x9d teaches a process for extracting and isolating SDG from de-fatted flaxseed that employs the following sequence of operations:
1.) De-fatted flaxseed meal is contacted with an xe2x80x9caliphatic alcohol solventxe2x80x9d (i.e. mixtures of methanol, ethanol, isopropanol, or butanol with water) to extract the SDG-containing lignan precursor.
2.) Residual solids are separated from the lignan-rich alcohol-water solvent and the lignan-containing extract is then concentrated by removing solvent by distillation until a syrup is obtained.
3.) SDG is liberated from its lignan precursor compounds by hydrolysis of the syrup at an elevated pH.
4.) The hydrolyzed aqueous concentrate is subjected to a liquid/liquid partition to further enrich the SDG. The preferred embodiment calls for the use of ethyl acetate to remove impurities that include the methyl esters of cinnamic acids and other cinnamic acid derivatives. Alternatively, the SDG-containing aqueous phase may be subjected to contact with an anion exchange resin to remove impurities.
5.) The aqueous hydrolysate is subjected to chromatographic separation using reverse-phase high-pressure liquid chromatography (xe2x80x9cHPLCxe2x80x9d) to isolate SDG.
U.S. Pat. No. 5,705,618 teaches the use of mixtures of aliphatic alcohols and water as the extraction solvent, with preference for alcohol-to-water ratios ranging from 1.85:1 to 3:1.
The present invention employs mixtures of acetone and water instead of aliphatic alcohols and water as the extraction solvent to extract SDG from de-fatted flaxseed.
In accordance with one aspect of the invention, a process for extracting secoisolariciresinol diglycoside (SDG) from de-fatted flaxseed is described. The process comprises contacting defatted flaxseed with an extraction solvent of acetone and water, extracting the de-fatted flaxseed with the extraction solvent to extract SDG-containing compounds, removal of solvents, followed by alkaline hydrolysis of the extract to liberate free SDG or its salts.
In accordance with another aspect of the present invention, a process for preparing a secoisolariciresinol diglycoside (SDG) concentrate from de-fatted flaxseed is disclosed. The process involves extracting defatted flaxseed with an extraction solvent of acetone and water to obtain an SDG-containing extract, separating residual solids from the SDG-containing extract, removing acetone to provide an acetone stripped extract, lowering the pH of the acetone stripped extract to precipitate SDG, separating precipitated SDG from the acetone stripped extract, subjecting the precipitated SDG in an aqueous slurry to hydrolysis to liberate free SDG and recovering the free SDG from the aqueous slurry.
In accordance with particular aspects of the invention, the precipitated SDG in the extract solution is subjected to hydrolysis to free the SDG from polymeric lignan precursor compounds. In accordance with particularly useful embodiments of the present invention, calcium hydroxide is used to liberate free SDG. The resulting SDG concentrates obtained using calcium hydroxide are non-hygroscopic and easily separated from insoluble calcium salts to provide a product of relatively high purity.
The present invention relates to an improved process for the extraction, isolation, and purification of secoisolariciresinol diglycoside (SDG) from de-fatted flaxseed. The process of the invention utilizes an extraction solvent comprising acetone and water to extract SDG from de-fatted flaxseed. The acetone-water solvent increases efficiency and selectivity of the extraction. Furthermore, the acetone can be easily removed after extraction and reused, thereby minimizing solvent usage.
The proportion of acetone to water will typically range from about 35% acetone/65% water by weight to about 65% acetone/35% water by weight. In accordance with one embodiment, the solvent contains about 45% acetone/55% water by weight.
The acetone-water solvent will typically be employed in a ratio of about 6 or more parts by weight solvent mixture to about 1 part by weight de-fatted flaxseed. Preferably, the solvent to feedstock ratio is in the range of about 12:1 to 16:1.
Although the extraction may be performed over a wide range of temperatures for various periods of time, in accordance with one embodiment, it is performed at acetone reflux for about 90 minutes with vigorous agitation.
The mixture is then filtered or centrifuged to remove the undissolved solids. Filtration or centrifugation is preferably performed at elevated temperatures, e.g., exceeding about 50xc2x0 C., to retain the SDG in solution.
About 18 to 20% of the mass of the feedstock may dissolve in the solvent. More than about 85% of the SDG-containing lignan precursor from the feedstock is generally contained in the extract from a single extraction.
Acetone is removed by distillation from the filtrate or centrifugate and recovered for re-use.
The acetone-water extraction solvent of the present invention enjoys several important advantages over the alcohol-water solvents of U.S. Pat. No. 5,705,618, notably:
1.) Improved efficiency of extraction per unit weight of solvent employed.
2.) Improved selectivity of extraction.
3.) Greater ease of solvent recovery. Unlike ethanol, isopropanol, and butanol, acetone does not azeotrope (i.e. form a constant boiling mixture) with water, making it easier to separate from the product-containing extract and recover for reuse.
4.) Acetone is far less toxic than methanol and emissions of acetone are not regulated under the Clean Air Act like those of methanol, ethanol, isopropanol, and butanol.
In accordance with certain embodiments of the present invention, calcium hydroxide may be used to liberate free SDG from the polymeric lignan precursor compounds that fall out of solution when acetone is removed from the de-fatted flaxseed extract by distillation. Sufficient calcium hydroxide is added to the aqueous slurry to achieve a pH of 10 to 13 (preferably 11.8 to 12.5), whereupon the resulting alkaline mixture is heated to approximately 50to 100xc2x0 C. (preferably to about 70 to 90xc2x0 C.) with agitation for a period of 30 minutes or more (preferably for a period of one to four hours) to effect the hydrolysis of the SDG-containing polymeric lignan precursor compounds.
After the alkaline hydrolysis is complete, the pH is lowered to a value of from about 6.5 to 8.5 (preferably to about 7.5) by adding phosphoric acid. Upon cooling, insoluble calcium phosphates (principally dibasic calcium phosphate or calcium monohydrogen phosphate; CaHPO4) fall out of solution and can be removed by filtration or centrifugation. The SDG remains in the filtrate or centrate.
One advantage of employing calcium hydroxide over alkalies of the Group I metals (i.e. sodium and potassium hydroxides or carbonates) is that dibasic calcium phosphate is only very sparingly soluble in water and can be readily separated from the post-hydrolysis SDG-containing aqueous solution by filtration or centrifugation. If sodium or potassium hydroxides or carbonates are employed, the sodium and potassium salts of phosphoric acid and other mineral acids (i.e., sulfuric acid, hydrochloric acid, etc . . . ) are highly soluble in the post-hydrolysis SDG-containing aqueous solution. Hence these salts cannot be readily separated from the SDG, resulting in a product of lower purity. Although these compounds provide lower purity, their use is still within the scope of the present invention which is not limited to any particular method for subjecting the SDG precipitate to hydrolysis.
A second advantage of employing calcium hydroxide is that the resulting SDG concentrates are not hygroscopic, unlike SDG concentrates prepared with sodium or potassium hydroxides or carbonates.
The SDG can be recovered by removing water by lyophilization, spray drying, vacuum drying, or other evaporative means to provide an SDG concentrate in a yield of at least about 70%, preferably at least about 85% and in accordance with certain embodiments as high as about 90% or greater.